Interferons are a family of cytokines with a wide variety of biological activities. They are components of the host defense against viral and parasitic infections and certain types of malignancies. They also affect the functioning of the immune system as well as cell proliferation and differentiation. The pleiotropic cellular effects of interferons are mediated by the interferon-inducible proteins. In this application, we propose to investigate the cellular functions and the structure-function relationships of two families of interferon-induced proteins: 2-5 (A) synthetases and the P56 family of proteins. 2-5 (A) Synthetases are a family of interferon-induced enzymes which require double-stranded RNA as an activator and synthesize 2'-5' linked oligoadenylates that activate the latent ribonuclease, RNase L. Production of recombinant 2-5 (A) synthetases in insect cells and their purification have recently been achieved by us. One objective of this project is to study the nature of the interaction between the recombinant enzymes and double-stranded RNA using methods recently standardized in our laboratory. Another objective is to study the role, in enzyme activity, of the putative active center and the post- translational modifications of the proteins. Finally, the noted differences in the antiviral and the anti-cellular activities of the medium and the small isozymes will be explored. These studies will required the use of genetic, biochemical and biophysical techniques. Another interferon-inducible family of proteins is represented by P56, the product of the 561 gene cloned by us. Using yeast two-hybrid screening, P56 has been shown to interact with the cellular protein, Int-6 and the human papilloma viral protein, E1. Int-6 is involved in cell growth regulation and its malfunction causes mammary carcinoma in mice. It is also a component of the translation initiation factor eIF3. The nature of p56-Int-6 interaction and how it affects the functions of Int-6 in protein synthesis and cell growth regulation will be investigated. The HPV E1 protein is absolutely required for viral DNA replication and it appears that P56 and interferon can inhibit HPV replication. The characteristics and the mechanism of this inhibition will be investigated as a part of this proposal. Whether this antiviral effect of P56 functions against many types of HPV will also be investigated. For these studies, advantage will be taken of an in vitro differentiation system of keratinocytes that allows HPV gene expression, replication and morphogenesis.